Animals may be protected against pestiviruses by vaccination, however, conventional inactivated or modified live vaccines have disadvantages concerning safety as well as efficacy. Therefore, new types of vaccines should be developed.
Pestiviruses can be divided into two different biotypes, cytopathogenic (cp) and non cytopathogenic (ncp) viruses, respectively. Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus within the family Flaviviridae is the causative agent of bovine viral diarrhea, an economically important disease of cattle world-wide. Genetically and structurally closely related virus species are Classical Swine Fever Virus (CSFV) and the ovine Border Disease Virus (BDV). Pestiviruses can induce severe diseases with marked economical losses world wide. The major economic losses caused by BVDV infections are reduced fertility, abortions and the generation of persistently infected calves, which can develop fatal “Mucosal Disease”. While cp BVDV induce apoptosis and cell death and express non-structural protein 3 (NS3), inoculation with ncp BVDV leads to persistent infection of cell cultures and NS3-expression is not detectable. The pestivirus genome consists of a single-stranded RNA of positive orientation. The RNA has a length of approximately 12.3 kb and contains one large open reading frame (ORF), which is flanked by non-translated regions (NTR) at both genome ends. The pestiviral ORF is translated into one polyprotein, which is co- and post-translationally processed into 11 (ncp BVDV) or 12 (cp BVDV) mature proteins by viral and cellular proteases. Pestivirus virions consist of four structural proteins, a capsid (C) protein and three glycosylated envelope proteins (ERNS, E1, E2). BVDV antibodies are directed against ERNS, E2 and NS3. Neutralizing activity was predominantly demonstrated for E2-specific antibodies.
Studies on the replication of pestiviruses have been considerably facilitated by reverse genetic systems and the discovery of autonomously replicating subgenomic RNAs (replicons) (4, 26).
Pestivirus genomes with deletions were first described as defective interfering particles (DI) for BVDV and CSFV (24, 27).
Since then many reports relating to the replication of pestiviruses have been published. Trans-complementation of structural proteins of viruses of the family Flaviviridae has been reported.
Pestivirus self-replicating RNAs are important tools for an understanding of virus replication, assembly and egress. Trans-complementation of deleted parts of the genome can be used, for example, for the identification of trans-acting elements of a pestiviral genome.
The minimal requirements for CSFV replication were investigated, for example, by creating defective CSFV genomes lacking the gene sequences for the structural proteins. It was found that the defective CSFV genomes still replicated and could be packaged into viral particles when introduced in SK-6 cells together with helper A187-CAT RNA (Moser et al., J. Virol., 7787-7794, 1999). An autonomously replicating defective BVDV genome which lacks the genes encoding C, Ems, E1, E2, p7 and NS2 had been described (Behrens et al., J. Virol., 72, 2364-2372, 1998). For the Kunjin virus (KUN) replicons with deletions in the structural regions were packaged into virons using a system where BHK-21 cells were transfected by two consecutive electropoartions, first with the mutated KUN replicons, and subsequently with a recombinant Semlike Forest Virus replicon expressing KUN structural proteins (Khromykh et al., J. Virol., 72(7), 5967-5977, 1998). For CSFV it had been recognized that trans-complemented defective virions that contained Ems deleted replicons could potentially serve as a basis for vaccines. CSFV, ERNS-deleted replicons were generated and trans-complemented using a swine kidney cell line (SK6) constitutively expressing CSFV ERNS. The resulting virions were able to infect SK6 cells without the production of infectious virus progeny and could be passaged on ERNS-expressing SK6 cells. Pigs were protected against lethal CSFV challenge after immunization with the complemented virions (40).
So far, there are only few reports concerning trans-complementation of BVDV replicons. Defects in the coding regions for non-structural proteins NS3, NSNS4a, NS4B and NS5B were found to be non complementable, whereas defects in the NS5A unit could be complemented in trans (Grassmann et al., J. Virol., 75, 7791-7802, 2001). (4, 18, 36) Trans-complementation of BVDV E2 and/or p7, using cell lines constitutively expressing BVDV E2 and/or p7 for complementation have also been described (Harada et al., J. Virol., 74, 9498-9506, 2000).